Increased cardiac Ang II activity from intracellular or interstitial formation (autocrine/paracrine mechanisms) is a cause of cardiac hypertrophic remodeling, arrhythmias, and fibrosis. RAS inhibitors are less effective in blocking Ang II-mediated adverse cardiac remodeling because its production within cardiac myocytes follows non-canonical pathways and the plasma membrane imposes a significant barrier to the external environment. The overarching hypothesis of this Program Project Grant is that the non-canonical pathway for intracellular cardiac Ang II production is mediated by a non-renin/ACE pathway. Cardiac chymase, incorporated into these cells from activated mast cells, acts on an endogenously shorter form of angiotensinogen (Aogen) -angiotensin-(1-12) [Ang-(1-12)] - to generate Ang II. The Project leaders' complementary clinical and basic science expertise in this renewal application will address this concept by examining the intracellular pathways and mechanisms accounting for the chymase-mediated Ang II formation and matrix metalloproteinase activation, the potential role of kallikrein as the Ang-(1-12) generating enzyme from Aogen, and the provocative new finding that estrogen deficiency, by modulating the cardiac intracellular chymase/Ang-(1-12) axis, contributes to the adverse cardiac remodeling and left ventricular diastolic dysfunction in postmenopausal women. The integrated research plan employs genetic models of well characterized altered cardiac function and structure (SHR and congenic mRen2.Lewis hypertensive rats; Projects 1 and 3), a cardiac myocyte cell line (HL-1) that maintains cardiac-specific phenotypes, and human left atrial tissue and cardiac pericardial fluid from subjects undergoing cardiac surgery for the correction of mitral regurgitation (Project 2). The additional novel inclusion of studies in rats expressing the human genes for Aogen, chymase, or both will eventually provide a translational laboratory paradigm for unraveling the cellular and biochemical mechanisms of the distinct biotransformation processes by which the human heart regulates production of Ang II in health and disease. Overall, the combined tightly focused efforts of Drs. Ferrario (Project 1), Dell'Italia (Project 2) and Groban (Project 3) will shed new light on the molecular mechanisms of cardiac Ang II contribution to adverse remodeling and progression to heart failure, the distinct ways by which human and rodent cardiac myocytes differ in terms of the cellular pathways for Ang II production, and how chymase and Ang-(1-12) are regulated by estrogen. These discoveries will identify new therapeutic approaches of higher efficacy when compared to angiotensin converting enzyme inhibitors and Ang II receptor blockers.